KR100763905B1 - Microfluidic device for electrochemically regulating the pH of a fluid therein using semiconductor doped with impurity and method for regulating the pH of a fluid in a microfluidic device using the same - Google Patents

Abstract

The present invention comprises: a) a cathode substrate; b) an anode substrate positioned opposite said cathode substrate and forming a reaction chamber with said cathode substrate; And c) an insulator separating the boundary between the cathode substrate and the anode substrate, wherein at least one of the cathode substrate and the anode substrate is a semiconductor doped with impurities and the remainder is a metal electrode electrochemically It provides a microfluidic device for adjusting the pH and a method for adjusting the pH using the same. The microfluidic device of the present invention does not require a separate electrode, and thus does not require an electrical connection between the electrode and the outside, thereby miniaturizing the microfluidic device, simplifying the manufacturing process, solving a leak of the chamber, and manufacturing cost. Can be reduced. In addition, the microfluidic device of the present invention can integrate various biological analysis processes including a microstructure capable of adsorbing biomolecule material inside the chamber of the cathode substrate, and can be easily manufactured using a semiconductor manufacturing process. . According to the method of the present invention, it is possible to quickly and easily adjust the pH of the fluid in the microfluidic device and / or to efficiently concentrate the biomolecule material.

Electrolysis, pH, Microfluidic Device, Lab-on-a-Chip, Doped Silicon, Microstructure, Cell Lysis

Description

Microfluidic device for electrochemically regulating the pH of a fluid therein using semiconductor doped with impurity and method for regulating the pH of a fluid in a microfluidic device using the same}

1 is a side cross-sectional view showing an example of a conventional microfluidic device for electrochemically adjusting the pH of a fluid.

Figure 2 is a side cross-sectional view showing an example of a microfluidic device according to the present invention.

3 is a side cross-sectional view showing another example of a microfluidic device according to the present invention.

Figure 4 is a side cross-sectional view showing another example of a microfluidic device according to the present invention.

Figure 5 is an enlarged side cross-sectional view of an example of the microstructure formed in the chamber of the cathode substrate of the microfluidic device according to the present invention.

Figure 6 is an enlarged top view of another example of the microstructure formed inside the chamber of the cathode substrate of the microfluidic device according to the present invention.

Figure 7 is a photograph showing a microfluidic device according to the present invention produced in the embodiment of the present invention.

8 is a graph measuring the amount of current according to the type of cathode and anode electrode of the microfluidic device according to the present invention for each voltage (A: anode (Pt) / cathode (Pt), B: anode (n-type Si)) / Cathode (n-type Si), C: anode (p-type Si) / cathode (p-type Si), D: anode (Pt) / cathode (n-type Si), E: anode (Pt) / cathode (p-type Si )).

Figure 9A is a graph showing the result of measuring the pH after the voltage application of the cathode chamber of the microfluidic device according to the present invention (A: anode (Pt) / cathode (Pt), B: anode (n-type Si) / cathode) (n-type Si), C: anode (p-type Si) / cathode (p-type Si), D: anode (Pt) / cathode (n-type Si), E: anode (Pt) / cathode (p-type Si)) .

Figure 9B is a graph showing the result of measuring the pH after voltage application of the anode chamber of the microfluidic device according to the present invention (A: anode (Pt) / cathode (Pt), B: anode (n-type Si) / cathode) (n-type Si), C: anode (p-type Si) / cathode (p-type Si), D: anode (Pt) / cathode (n-type Si), E: anode (Pt) / cathode (p-type Si)) .

Figure 10 is a graph measuring the DNA stability in the cathode according to the type of cathode electrode of the microfluidic device according to the present invention.

11 is a graph measuring cell solubility in a cathode chamber according to the type of cathode and anode electrode of the microfluidic device according to the present invention.

The present invention relates to a microfluidic device for electrochemically adjusting the pH of a fluid and a method for electrochemically adjusting the pH of a fluid in a microfluidic device using the same.

The microfluidic device refers to a device in which the inlet, the outlet, and the reaction vessel are fluidly connected through the microchannel. In addition to the microchannel, the microfluidic device is generally provided with a micropump for transporting a fluid, a micromixer for mixing a fluid, and a microfilter for filtering the transported fluid.

Such microfluidic devices are well known in the art and include cell enrichment in samples, cell lysis, biomolecular purification, amplification and isolation of nucleic acids such as PCR, protein isolation, hybridization, and detection, It is used in microanalytical devices such as lab-on-a-chip (LOC) that perform the same series of biological analyses.

In order to perform such various biological analytical procedures using a microfluidic device, a different pH is required for each step. In the biological analysis process, conventional pH adjustment was made by adding or removing acidic solution, basic solution, neutral solution or buffer solution. However, in the case of adding or removing such a pH adjusting solution in the microfluidic device, not only a separate device and a procedure are required, but also a problem in that the sample solution is diluted. These injection step and device problems can be a serious problem for microfluidics handling microvolumes, and dilution can be a problem when taking or amplifying the desired sample. In addition, the pH-adjusting substance thus added may also be removed after use if it can act as an inhibitor in a subsequent biological analysis.

There is a method using the electrolysis as a method for solving the problems of the prior art injecting the pH control reagent from the outside. For example, there is a method of adjusting pH by using an electrolysis device including an anode chamber, a cathode chamber, and a partition membrane installed between the chambers. 1 is a side cross-sectional view showing an example of a conventional microfluidic device for electrochemically adjusting the pH of a fluid. Referring to FIG. 1, the conventional apparatus includes a cathode 11, an anode 13 and a partition film 15 blocking therebetween. In addition, the prior art device must pull the conductor out of the reaction chamber to electrically connect the cathode 11 and the anode 13 to the power supply 17. However, such electrical connection acts as a limit to the miniaturization of the microfluidic device, complicates the manufacturing process, and increases the manufacturing cost.

Meanwhile, microstructures capable of adsorbing or binding to cells are known in the art. For example, the microstructures that can adsorb or bind the cells are porous, pillar, and sieve structures (ANALYTICAL BIOCHEMISTRY 257, 95-100 (1998), Integrated Cell Isolation and Polymerase Chain Reaction Analysis). Using Silicon Microfilter Chambers, Peter Wilding; see US 6,440,725.

In microfluidic devices used in lab-on-a-chip, the integration of each configuration and function is an important goal for the automation of all analytical processes. However, there have been no attempts to integrate the pH control function and the cell adsorption or binding function.

The present invention has been made to solve the problems of the prior art, it is an object of the present invention to provide a microfluidic device for electrochemically controlling the pH of a fluid.

Another object of the present invention is to provide a method of controlling the pH of a fluid in a microfluidic device by electrolysis using the microfluidic device.

In order to achieve the object of the present invention, the present invention comprises: a) a cathode substrate; b) an anode substrate positioned opposite said cathode substrate and forming a reaction chamber with said cathode substrate; And c) an insulator separating the boundary between the cathode substrate and the anode substrate, wherein at least one of the cathode substrate and the anode substrate is a semiconductor doped with impurities and the remainder is a metal electrode electrochemically It provides a microfluidic device for adjusting the.

The microfluidic device according to the invention can be used for cell lysis. The microfluidic device for cell lysis includes a) a cathode substrate which is a semiconductor doped with impurities; b) an anode substrate positioned opposite the cathode substrate and forming a reaction chamber together with the cathode substrate and being a semiconductor or metal electrode doped with impurities; And c) an insulator separating the boundary between the cathode substrate and the anode substrate, wherein a microstructure capable of adsorbing the biomolecule material is formed inside the chamber of the cathode substrate.

In order to achieve another object of the present invention, one embodiment of the present invention comprises a) ions having a lower standard oxidation potential or higher ions than water containing ions in the reaction chamber, and ions having a lower standard reduction potential than water. Introducing a solution; And b) applying a voltage through the anode substrate and the cathode substrate to cause electrolysis in the reaction chamber to adjust the pH of the solution introduced into the reaction chamber. It provides a method of electrochemical control.

In order to achieve another object of the present invention, another embodiment of the present invention comprises the steps of: a) introducing a solution containing a high ion or a low ion oxidation potential than the water in the anode chamber; b) introducing a solution containing ions having a lower standard reduction potential than water into the cathode chamber; And c) applying a voltage through an anode substrate and a cathode substrate to cause electrolysis in the anode chamber and the cathode chamber to adjust the pH of the solution introduced into the anode chamber or the cathode chamber, according to the present invention. A method of electrochemically controlling the pH of a fluid in a device is provided.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

One aspect of the invention relates to a microfluidic device for electrochemically adjusting the pH of a fluid.

Figure 2 is a side cross-sectional view showing an example of a microfluidic device according to the present invention.

2, the microfluidic device for electrochemically adjusting the pH according to the present invention comprises: a) a cathode substrate 101; b) an anode substrate (103) positioned opposite the cathode substrate (101) and forming a reaction chamber together with the cathode substrate (101); And c) a nonconductor 105 that separates the boundary between the cathode substrate 101 and the anode substrate 103, wherein at least one of the cathode substrate 101 and the anode substrate 103 is a semiconductor doped with an impurity. And the rest are metal electrodes.

The cathode substrate 101 and the anode substrate 103 are electrically connected to an external power supply 109.

Referring back to FIG. 2, the cathode substrate 101 or the anode substrate 103 made of a semiconductor doped with impurities of a microfluidic device for electrochemically adjusting pH according to the present invention itself serves as an electrode. As a result, it does not require a separate electrode, thereby having the advantage of not requiring an electrical connection between the electrode inside the chamber and the external power supply. Therefore, the configuration as described above can reduce the size of the microfluidic device, simplify the manufacturing process, solve the leakage problem of the chamber, and reduce the manufacturing cost.

The cathode substrate 101 or the anode substrate 103 made of the semiconductor doped with the impurity may be a group 14 element of the periodic table doped with an impurity, and the impurity may be a group 13 element or a group 15 element of the periodic table. In particular, the cathode substrate 101 or the anode substrate 103 made of the semiconductor doped with the impurity may be n-type silicon or p-type silicon.

3 is a side cross-sectional view showing another example of a microfluidic device according to the present invention.

Referring to FIG. 3, the microfluidic device of FIG. 3 has a different configuration from that of the microfluidic device of FIG. 2 and the anode substrate 103, and accordingly, an electrical connection form with the external power supply device 109 is different. That is, the anode substrate 103 may be an insulator including an anode 111 selected from the group consisting of platinum, gold, copper, aluminum, palladium, and titanium inside the chamber. When the copper electrode is used as the anode, the formation of CuCl ₂ when the chlorine ion such as NaCl is included in the anode chamber can reduce generation of chlorine gas which is a poison gas.

In contrast to FIG. 3, the anode substrate is made of a semiconductor doped with impurities, and the cathode substrate may be an insulator including a cathode selected from the group consisting of platinum, gold, copper, aluminum, palladium and titanium inside its chamber. (Not shown). When the palladium electrode is used as the cathode, the gas removal process is unnecessary because it absorbs hydrogen gas generated in the cathode chamber.

 Figure 4 is a side cross-sectional view showing another example of a microfluidic device according to the present invention.

Referring to FIG. 4, the microfluidic device of FIG. 4 differs from the microfluidic device of FIG. 2 in that its reaction chamber includes an ion exchange membrane 113, whereby the reaction chamber is divided into a cathode chamber and an anode chamber. Do.

In the device of the present invention, the reaction chamber, the anode chamber and the cathode chamber refer to a space capable of accommodating a material such as a fluid. It is not limited to. The chamber may be any one or more selected from the group consisting of a cell concentration chamber, a cell lysis chamber, a nucleic acid separation / purification chamber, a nucleic acid amplification chamber, a hybridization chamber, and a signal detection chamber. The chamber may be connected to various other chambers through microchannels. Therefore, the microfluidic device of the present invention may be in the form of a lab-on-a-chip capable of electrochemically adjusting the pH of the fluid.

When the chamber is in particular a cell lysis chamber, the microfluidic device according to the invention comprises: a) a cathode substrate which is a semiconductor doped with impurities; b) an anode substrate positioned opposite the cathode substrate and forming a reaction chamber together with the cathode substrate and being a semiconductor or metal electrode doped with impurities; And c) an insulator separating a boundary between the cathode substrate and the anode substrate, and a microstructure capable of adsorbing the biomolecule material inside the chamber of the cathode substrate may be formed.

In the present invention, the ion-exchangeable material has the property of passing current but not passing ions and / or gases generated by electrolysis in each chamber. Preferably, the ion exchanger has the property of passing a current but not of hydrogen and hydroxide ions and / or gases.

The ion exchange material may be a cation exchange membrane or an anion exchange membrane.

In the present invention, the metal ion exchange membrane may be an alkali metal ion exchange membrane. The cation exchange membrane is a membrane that passes through cations but exhibits a resistance close to 100%. The anion exchange membrane is a membrane that exhibits resistance close to 100% through a cation but passes through anions. For example, the cation exchange membrane may be a strong acid exchange membrane (containing -SO _3- ; Nafion) or a weak acid exchange membrane (containing -COO-), and the anion exchange membrane is a strong base exchange membrane (strong base) N ⁺ (CH ₃ )) or a weak base (including N (CH ₃ ) ₂ ). The cation exchange membrane and the anion exchange membrane are well known in the art, and those skilled in the art will be readily able to purchase and use them. For example, the ion exchange membrane is commercially available from Nafion ^™ (Dupont), Dowex ^™ (Aldrich), Diaion ^™ (Aldrich) and the like.

In one embodiment of the present invention, the microfluidic device of the present invention may be introduced into the reaction chamber or the anode chamber a solution containing ions or high ions having a lower standard oxidation potential than water, that is, an electrolyte that is electrolyzed. The ion having a lower standard oxidation potential than the water may be one or more ions including anion such as NO ₃ ⁻ , F ⁻ , SO ₄ ^{2 −} , PO ₄ ^{3 −,} and CO ₃ ^{2 −} , and may have a standard oxidation potential higher than that of water. High ions may be an electrolyte containing Cl ⁻ ions, but are not limited to these examples. When the reaction chamber or the anode chamber solution is a compound having a lower standard oxidation potential than water, when electrolysis is performed using a microfluidic device according to an embodiment of the present invention, water is reacted at the anode electrode of the reaction chamber or the anode chamber. This electrolysis produces oxygen gas and H ⁺ ions. In this case, the pH of the anode chamber or the anode chamber solution of the reaction chamber is lowered by the H ⁺ ions. Cl ⁻ ions, which have a higher standard oxidation potential than water, may be used specifically for the purpose of cell lysis only.

In addition, in another embodiment of the present invention, the microfluidic device of the present invention may be introduced into the reaction chamber or the cathode chamber containing a solution containing ions having a lower standard reduction potential than water. Examples of such ions may be a ^{Na +, K +, Ca 2} +, Mg 2 +, and Al ^{3 +} cations such as, but is not limited to these examples. Therefore, when electrolysis is performed using the microfluidic device according to the embodiment of the present invention, water is electrolyzed at the cathode of the reaction chamber or the cathode chamber to generate hydrogen gas and OH ⁻ ions. In this case, the pH near the anode electrode or the cathode chamber solution of the reaction chamber is increased by the OH ⁻ ions.

2 to 4, the microfluidic device for electrochemically adjusting pH according to the present invention has a microstructure capable of adsorbing biomolecule material inside the chamber of the cathode substrate 101 or the anode substrate 103. 107 may be formed.

The biomolecule material may be selected from the group consisting of DNA, RNA, peptides, proteins, bacteria and viruses.

The microstructure 107 capable of adsorbing the biomolecule material may be selected from the group consisting of a porous structure, a pillar structure, and a sieve structure.

5 is an enlarged side cross-sectional view showing an example of a microstructure formed inside the chamber of the cathode substrate of the microfluidic device according to the present invention, and FIG. 6 is formed inside the chamber of the cathode substrate of the microfluidic device according to the present invention. It is the top view which expanded the other example of the fine structure.

5 and 6, it can be seen that the microstructures 107 that can adsorb the biomolecule material are pillar structures 115 and 115 ′.

The microfluidic device according to the present invention including the microstructure 107 capable of adsorbing the biomolecule material has the advantage of integrating the pH control function and the concentration function of the biomolecule material in the chamber.

In addition, the microstructure 107 that can adsorb the biomolecule material has an advantage that it can be easily formed inside the chamber of the cathode substrate 101 using a conventional semiconductor manufacturing process.

In the present invention, the cathode substrate or the anode substrate may further include a gas outlet. For example, oxygen gas or hydrogen gas may be discharged out of the chamber through the gas outlet.

In the present invention, the anode chamber and the cathode chamber may further include inlets and outlets through which the solution enters and exits, respectively. The inlet and outlet do not necessarily have to be provided separately and one port may serve as an inlet and an outlet. The gas outlet may also be utilized as an inlet and / or outlet.

In the present invention, the anode chamber and the cathode chamber may each further include a pump for introducing and discharging the solution.

Another aspect of the present invention provides a method of controlling the pH of a fluid in a microfluidic device by electrolysis using the microfluidic device according to the present invention.

A method of adjusting the pH of a fluid in a microfluidic device by electrolysis using an embodiment of the present invention wherein the reaction chamber does not include an ion exchange membrane (see, for example, FIG. 2 or FIG. 3) is a) ion in the reaction chamber. Introducing a solution containing ions having a lower standard oxidation potential or higher ions than water, and ions having a lower standard reduction potential than water; And b) applying a voltage through the anode substrate and the cathode substrate to cause electrolysis in the reaction chamber to adjust the pH of the solution introduced into the reaction chamber.

A method of adjusting the pH of a fluid in a microfluidic device by electrolysis using an embodiment of the present invention wherein the reaction chamber comprises an ion exchange membrane (see, for example, FIG. 4) comprises: a) standard oxidation potential over water in the anode chamber; Introducing a solution containing low ions or high ions; b) introducing a solution containing ions having a lower standard reduction potential than water into the cathode chamber; And c) applying a voltage through the anode substrate and the cathode substrate to cause electrolysis in the anode chamber and the cathode chamber to adjust the pH of the solution introduced into the anode chamber or cathode chamber.

In the method of the present invention, the solution includes the biomolecule material, and the biomolecule material is adsorbed and concentrated in a microstructure capable of adsorbing the biomolecule material formed inside the chamber of the cathode substrate before the voltage application step. It may further comprise the step. In the case of carrying out this step, the pH of the solution around the adsorbed biomolecule material may be sufficiently adjusted, especially if the reaction chamber does not comprise an ion exchange membrane.

In the method of the present invention, examples of the anion having a standard oxidation potential lower than water, an anion having a standard oxidation potential higher than water, and a cation having a standard reduction potential lower than water are as described above.

The pH may be adjusted by the direction of the applied voltage, the intensity of the applied voltage, the voltage application time, the width of the electrode or the interval between chambers. The exact direction of the applied voltage, the intensity of the applied voltage, the voltage applied time, the area of the electrode and the inter-chamber spacing may vary depending on the desired pH or the volume of the chamber, etc., which can be easily determined by experimentation by those skilled in the art.

When the sample solution containing NaCl, which is the most contained in the biosample solution, is introduced into the anode and cathode and then electrolyzed, chlorine ions, not water, are electrolyzed at the anode to generate chlorine gas, which is less than the hydroxide ions generated at the cathode. A large amount of hydrogen ions are generated and this amount is caused by the reaction between chlorine gas and water, and it is difficult to adjust pH because it depends on the dissolution condition of chlorine gas. In the present invention, in order to solve this problem, a compound having a lower standard oxidation potential than water and / or a compound having a lower standard reduction potential than water is used in the reaction chamber or the anode chamber and the cathode chamber. However, in case of lysis only, the sample solution containing NaCl may be introduced into the reaction chamber, the anode chamber and the cathode chamber, followed by electrolysis to dissolve the cells at the cathode.

In the method of the present invention, since the cathode contains a reaction chamber or a cathode chamber solution containing a compound having a lower standard reduction potential than water, water is electrolyzed to generate hydrogen gas and OH ⁻ ions. In addition, since the anode contains a reaction chamber or an anode chamber solution containing a compound having a lower standard reduction potential than water, water is electrolyzed to generate oxygen gas and H ⁺ ions. As a result, the cathode solution has a basic pH and the anode solution has an acidic pH.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

<Example 1>

Preparation of microfluidic device for pH adjustment according to the present invention

A cathode substrate made of silicon doped with an impurity; An anode substrate disposed opposite the cathode substrate and doped with impurities to form a reaction chamber with the cathode substrate; And an insulator separating a boundary between the cathode substrate and the anode substrate, wherein the reaction chamber includes an ion exchange membrane, whereby the reaction chamber is divided into a cathode chamber and an anode chamber, and is formed inside the chamber of the cathode substrate. A microfluidic device for electrochemically controlling the pH of a fluid, characterized in that a fine pillar structure capable of adsorbing molecular material is formed.

Specifically, each volume of the cathode chamber and the anode chamber was 10 μl. In addition, a cation exchange membrane containing a -SO ₃ -Na ⁺ group was used as the ion exchange membrane. As the cathode substrate and the anode substrate, n-type silicon doped with arsenic (As), respectively, (resistance <0.005 kcm) was used. The chamber interior of the cathode substrate and anode substrate had dimensions of 2 mm x 3 mm, respectively.

Figure 7 is a photograph showing a microfluidic device according to the present invention produced in this embodiment.

<Example 2>

Preparation of microfluidic device for pH adjustment according to the present invention

The same microfluidic device as in Example 1 was prepared except that p-type silicon (resistance <0.005 kcm) doped with boron (B) was used as the cathode substrate and the anode substrate, respectively.

<Example 3>

Preparation of microfluidic device for pH adjustment according to the present invention

Except for using p-type silicon doped with boron (B) as the cathode substrate (resistance <0.005 kcm) and n-type silicon doped with arsenic (As) as the anode substrate (resistance <0.005 kcm) Manufactured the same microfluidic device as in Example 1.

<Example 4>

Preparation of microfluidic device for pH adjustment according to the present invention

Except for using n-type silicon doped with arsenic (resistance <0.005 kcm) as the cathode substrate and p-type silicon doped with boron (B) (resistance <0.005 kcm) as the anode substrate Manufactured the same microfluidic device as in Example 1.

<Example 5>

Preparation of microfluidic device for pH adjustment according to the present invention

Except for using an n-type silicon doped with arsenic (As) as a cathode substrate (resistance <0.005 kcm) and an insulator containing 2 mm x 3 mm of platinum (Pt) inside the chamber as an anode substrate. Manufactured the same microfluidic device as in Example 1.

<Example 6>

Preparation of microfluidic device for pH adjustment according to the present invention

Except for using p-type silicon doped with boron (B) (cathode resistivity <0.005 kcm) as the cathode substrate and an insulator containing 2 mm x 3 mm of platinum (Pt) inside the chamber as the anode substrate. Manufactured the same microfluidic device as in Example 1.

Experimental Example 1

Current intensity measurement of applied voltage of the microfluidic device according to the present invention

Using the microfluidic device according to the present invention prepared in Examples 1, 2, 5 and 6 was measured the current intensity for a constant applied voltage. The current strength is proportional to the pH change.

That is, the cathode chamber and the anode chamber of the microfluidic device according to the present invention prepared in Examples 1, 2, 5 and 6 were respectively filled with 55 mM Na ₂ SO ₄ aqueous solution, and 5 V, 7 V, and 9 V, respectively, at room temperature. And a DC voltage of 12 V was applied to measure the current between both electrodes.

8 is a graph measuring the amount of current according to the type of cathode and anode of the microfluidic device according to the present invention for each voltage. In Fig. 8, each A is a control microfluidic device (anode (Pt) / cathode (Pt)), B is a microfluidic device of Example 1 (anode (n-type Si) / cathode (n-type Si)), C is the microfluidic device of Example 5 (anode (p-type Si) / cathode (p-type Si)), D is the microfluidic device of Example 2 (anode (Pt) / cathode (n-type Si)), and E is The result of the microfluidic device of Example 6 (anode (Pt) / cathode (p-type Si)).

The current intensity was performed using a current measuring device (Agilent E3620A Dual output DC power supply). Since the device is a device measured in 1 mA units, currents with values below that are all expressed as 0 mA.

As can be seen in FIG. 8, when a voltage of 7 V or more is applied, a current flows between the electrodes of the microfluidic devices of Examples 5 and 6, and when a voltage of 9 V or more is applied, Example 1 and Example An electric current flowed between the electrodes of the microfluidic device of 2.

Therefore, although there was a slight difference as described above, it can be seen that all the microfluidic devices prepared in Examples 1, 2, 5, and 6 exhibit sufficient current strength, and thus can be effectively used for pH control through electrolysis.

Experimental Example 2

PH change measurement of the microfluidic device according to the present invention

The pH change of the chamber was measured using the microfluidic device according to the present invention prepared in Examples 1, 2, 5 and 6.

That is, the cathode and anode chambers of the microfluidic devices according to the present invention prepared in Examples 1, 2, 5 and 6 were respectively filled with 55 mM Na ₂ SO ₄ aqueous solution, and 5 V and 7 V, respectively, at room temperature for 40 seconds. DC voltages of 9 V and 12 V were applied to measure the pH of the cathode and anode chambers. The initial value was pH 7.

The results are shown in FIGS. 9A and 9B. 9A is a graph illustrating a result of measuring pH after voltage application of a cathode chamber of a microfluidic device according to the present invention, and FIG. 9B is a result of measuring pH after voltage application of an anode chamber of a microfluidic device according to the present invention. Is a graph. 9A and 11B, A, B, C, D and E are the same as in FIG.

9A, the pH of the cathode chamber was rapidly increased from 7.0 to 8.0 to 14.0 by the application of a voltage of 5 to 12 V. In particular, a voltage of more than 12 V required for cell lysis can be ensured by humans having a voltage of 7 V or higher. there was. Referring to FIG. 9B, the pH of the anode chamber dropped sharply from 7.0 to about 1.0 to 5.5 by applying a voltage of 5 V to 12 V. The graph of B, which is the result of the microfluidic device of Example 1 (anode (n-type Si) / cathode (n-type Si)), shows that in the anode chamber, unlike in the cathode chamber, there is almost no pH change and almost no bubbles are generated. I didn't see it.

From the above results, it can be seen that all the microfluidic devices prepared in Examples 1, 2, 5 and 6, in particular the microfluidic devices of Examples 5 and 6, can very effectively adjust the pH of the solution in the chamber.

Experimental Example 3

DNA stability test in microfluidic device according to the present invention

DNA stability at the cathode of the microfluidic device prepared in Example, ie, the degree of adsorption of DNA was compared.

First, the cathode chamber and the anode chamber of the microfluidic devices of Examples 5 and 6 were respectively filled with 55 mM Na ₂ SO ₄ , and the culture of E. coli (BL21, Stratagen) was respectively filled with 5 × 10 ⁴ copies / It was added in the amount of chamber. Next, electrolysis was performed by applying a DC voltage of 5 V at room temperature for 40 seconds.

The use of platinum as a cathode and the use of DNA without electrolysis were compared as controls.

Quantitative PCR was performed on the solution obtained by the above procedure as a template to measure the extent to which the cells were dissolved and adsorbed onto the cathode. Forward primers and reverse primers were used as primers (FP: 5 'YCCAKACTCCTACGGGAGGC 3' (SEQ ID NO: 1), RP: 5 'GTATTACCGCRRCTGCTGGCAC 3' (SEQ ID NO: 2)).

The amount of DNA was determined by Cp (crossing point) by quantitative PCR. Smaller Cp means higher initial DNA amount.

10 is a graph measuring the DNA stability in the cathode according to the type of cathode electrode of the microfluidic device according to the present invention.

As shown in Figure 10, the Cp value of the microfluidic device of the present invention was not significantly different compared to the control. From the above results, it can be seen that the stability of the DNA is good when using the microfluidic device according to the present invention.

Experimental Example 4

Cell lysis experiment using microfluidic device according to the present invention

Cell lysis experiments, one of a series of biological assays, were performed using the microfluidic device prepared in Example.

First, the cathode chamber and the anode chamber of the microfluidic devices of Examples 1 to 4 were respectively filled with 55 mM Na ₂ SO ₄ , and the culture of E. coli (BL21, Stratagen) was added to the cathode chamber in an amount of 10 ⁵ cells / chamber. Was added. The use of platinum as a cathode and an anode respectively and the case without electrolysis were compared as controls.

By applying a DC voltage of 5 V to the microfluidic devices of Examples 1 to 4, and a DC voltage of 5 V and 9 V to the microfluidic device using platinum as the cathode and anode, respectively, for 40 seconds at room temperature. Electrolysis was performed.

Quantitative PCR was performed on the solution obtained by the above procedure as a template to measure the extent to which the cells were dissolved and adsorbed onto the cathode. Forward primers and reverse primers were used as primers (FP: 5 'YCCAKACTCCTACGGGAGGC 3' (SEQ ID NO: 1), RP: 5 'GTATTACCGCRRCTGCTGGCAC 3' (SEQ ID NO: 2)).

The amount of DNA was determined by Cp (crossing point) by quantitative PCR. Smaller Cp (meaning the cycle in which the signal goes up) means more initial DNA. This is because the detection by the detector is quicker only when the initial amount is large. 11 is a graph measuring cell solubility in a cathode chamber according to the type of cathode and anode electrode of the microfluidic device according to the present invention.

As shown in Figure 11, the Cp value of the microfluidic device of the present invention was almost similar to the control. From the above results, it can be seen that when the microfluidic device according to the present invention is used, pH adjustment of the chamber can be effectively performed, whereby a series of biological analytical processes requiring different pH, such as cell lysis, can be effectively performed. Can be.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

The microfluidic device of the present invention does not require a separate electrode, and thus does not require an electrical connection between the electrode and the outside, thereby miniaturizing the microfluidic device, simplifying the manufacturing process, solving a leak of the chamber, and manufacturing cost. Can be reduced. In addition, the microfluidic device of the present invention can integrate various biological analysis processes including a microstructure capable of adsorbing biomolecule material inside the chamber of the cathode substrate, and can be easily manufactured using a semiconductor manufacturing process. . According to the method of the present invention, it is possible to quickly and easily adjust the pH of the fluid in the microfluidic device and / or to efficiently concentrate the biomolecule material.

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Claims (23)

a) a cathode substrate; b) an anode substrate positioned opposite said cathode substrate and forming a reaction chamber with said cathode substrate; And c) an insulator separating the boundary of the cathode substrate and the anode substrate, At least one of the cathode substrate and the anode substrate is a semiconductor and the remaining metal electrodes are microfluidic devices for electrochemically controlling the pH of a fluid. The method of claim 1, A microfluidic device, wherein an adsorption portion capable of adsorbing biomolecules is formed inside a chamber of the cathode substrate or the anode substrate, which is a semiconductor. a) a cathode substrate formed of a semiconductor; b) an anode substrate positioned opposite said cathode substrate and forming a reaction chamber together with said cathode substrate and being a semiconductor or metal electrode; And c) an insulator separating the boundary of the cathode substrate and the anode substrate, Cell lysis microfluidic device is characterized in that the adsorption portion for adsorbing biomolecules is formed inside the chamber of the cathode substrate. The method of claim 2 or 3, The biomolecule is a microfluidic device, characterized in that selected from the group consisting of DNA, RNA, peptides, proteins, bacteria and viruses. The method of claim 2 or 3, The adsorption unit is a microfluidic device, characterized in that selected from the group consisting of a pillar structure, a sieve structure, and a porous structure. The method according to claim 1 or 3, And the semiconductor is n-type silicon or p-type silicon. The method according to claim 1 or 3, The metal electrode is selected from the group consisting of platinum, gold, copper, aluminum, palladium and titanium. The method according to claim 1 or 3, The cathode substrate or the anode substrate further comprises a gas outlet. The method according to claim 1 or 3, The cathode substrate or the anode substrate each further comprises a microfluidic device further comprises an inlet and an outlet through which the solution is introduced and exited. The method according to claim 1 or 3, The anode substrate and the cathode substrate each microfluidic device further comprises a micropump for inflow and outflow of the solution. The method according to any one of claims 1 to 3, The reaction chamber comprises an ion exchange membrane, whereby the reaction chamber is divided into a cathode chamber and an anode chamber. a) introducing into the reaction chamber a solution comprising ions having a lower standard oxidation potential or higher ions than water containing ions and ions having a lower standard reduction potential than water; And b) applying a voltage through an anode substrate and a cathode substrate to cause electrolysis in the reaction chamber to adjust the pH of the solution introduced into the reaction chamber; A method of electrochemically adjusting the pH of a fluid in the microfluidic device according to any one of claims 1 to 3. a) introducing a solution containing ions having a lower standard oxidation potential or higher ions than water into the anode chamber; b) introducing a solution containing ions having a lower standard reduction potential than water into the cathode chamber; And c) applying a voltage through an anode substrate and a cathode substrate to cause electrolysis in the anode chamber and the cathode chamber to adjust the pH of the solution introduced into the anode chamber or cathode chamber; Method of electrochemically adjusting the pH of the fluid in the microfluidic device according to claim 11 comprising a. The method of claim 12, The solution comprises biomolecules, and further comprising adsorbing and concentrating the biomolecules on the adsorption portion formed in the semiconductor substrate before the voltage application step. The method of claim 12, The ion having a lower standard oxidation potential than water is at least one selected from the group consisting of NO ₃ ⁻ , F ⁻ , SO ₄ ^2- , PO ₄ ^3-, and CO ₃ ^2- . The method of claim 12, The ion having a higher standard oxidation potential than water is Cl ⁻ . The method of claim 12, The ion having a lower standard reduction potential than that of water is at least one selected from the group consisting of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ and Al ³⁺ . The method of claim 12, The pH is controlled by one or more of the direction of the applied voltage, the strength of the applied voltage, the voltage application time, the width of the electrode and the inter-chamber spacing. The method of claim 13, The solution comprises biomolecules, and further comprising adsorbing and concentrating the biomolecules on the adsorption portion formed in the semiconductor substrate before the voltage application step. The method of claim 13, The ion having a lower standard oxidation potential than water is at least one selected from the group consisting of NO ₃ ⁻ , F ⁻ , SO ₄ ^2- , PO ₄ ^3-, and CO ₃ ^2- . The method of claim 13, The ion having a higher standard oxidation potential than water is Cl ⁻ . The method of claim 13, The ion having a lower standard reduction potential than that of water is at least one selected from the group consisting of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ and Al ³⁺ . The method of claim 13, The pH is controlled by one or more of the direction of the applied voltage, the strength of the applied voltage, the voltage application time, the width of the electrode and the inter-chamber spacing.

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